The broad, long-term objectives of this proposal are: (1) Identify nuclear genes which can shed light on the tissue specificity and penetrance of mitochondrial related hearing loss; (2) identify candidate nuclear genes which may be responsible for autosomal recessive, autosomal dominant and/or x-linked inherited hearing loss; and (3) provide the basis for understanding the cellular machinery involved in mitochondrial RNA processing and translation. The specific aims are: (1) Identify nuclear genes whose products have a role in mitochondrial RNA processing and translation; (2) characterize the identified genes by (a) mapping their chromosomal location and comparing it to the known deafness loci, and (b) checking each one for isoforms or differential splicing in the cochlea; and (3) screen genes, which by chromosomal position overlap known deafness loci or have cochlea specific isoforms/splice variants, for mutations in appropriate patients. The health-relatedness of the project is in its ability to identify genes which lead to inherited hearing loss and influence the clinical expression of the hearing impairment. The experimental design and methodology will include the use of homology cloning, the yeast dihybrid method, and mitochondrial ribosomal protein extraction and microsequencing for the identification of genes involved in mitochondrial RNA processing and translation. These genes will be characterized for chromosomal location by testing radiation hybrid cell lines with gene specific amplification. Cochlea specific splicing and isoforms will be searched for by screening databases and cochlear cDNA libraries, Southern blotting, and direct protein comparisons. Mutational analysis of candidate genes will be done in the appropriate patient populations with standard molecular techniques and for nucleic acid analysis, the precise choice dependent on the tissue available for analysis and the presumed molecular defect.